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At least 1,243 records · Page 69Linked to original sources
PubMed2004

Simultaneous monitoring of Zn2+ secretion and intracellular Ca2+ from islets and islet cells by fluorescence microscopy.

A method for simultaneously imaging Zn2+ secretion and intracellular Ca2+ at beta-cell clusters and single islets of Langerhans was developed. Cells were loaded with the Ca2+ indicator Fura Red, incubated in buffer containing the Zn2+ indicator FluoZin-3, and imaged via laser scanning fluorescence confocal microscopy. FluoZin-3 and Fura Red are excited at 488 nm and emit at 515 and 665 nm, respectively. Zn2+, which is co-released with insulin, reacts with extracellular FluoZin-3 to form a fluorescent product. Stimulation of cell clusters with glucose evoked increases and oscillations in intracellular Ca2+ and Zn2+ secretion that were correlated with each other and were synchronized among cells. In single islets, spatially resolved dynamics of secretion including detection of first phase, second phase, and synchronized oscillations around the islet were observed. Fura Red did not yield detectable Ca2+ signals at islets. For islet measurements, cells were loaded with Fura-2 and incubated in FluoZin-3 while sequentially illuminating the islets with 340, 380, and 470 nm light and acquiring epi-fluorescence images with a charge-coupled device (CCD) camera. This allowed Ca2+ and secretion to be observed with approximately 2 s temporal resolution. This method should be useful for studying Ca2+ secretion coupling and any application, requiring rapid assays of secretion.

Animals↗
PubMed1997

Measurement of nucleotide release kinetics in single skeletal muscle myofibrils during isometric and isovelocity contractions using fluorescence microscopy.

Rabbit psoas muscle myofibrils, in the presence of the fluorescent nucleotide analog 2'(3')-O-[N-[2-[[Cy3]amido]ethyl]carbamoyl]-adenosine 5' triphosphate (Cy3-EDA-ATP), showed selective fluorescence staining of the A-band with a reduced fluorescence at the M-line. Addition of Cy3-EDA-ATP to a myofibril in the presence of Ca2+ caused auxotonic shortening against a compliant glass microneedle. These results indicate that Cy3-EDA-ATP is a substrate for myosin in the myofibril system. The kinetics of nucleotide release from a single myofibril, held isometrically between two needles, were measured by the displacement of prebound Cy3-EDA-ATP on flash photolysis of caged ATP. The A-band fluorescence of the myofibril decayed exponentially with a rate constant of 0.3 s(-1) at 8 degrees C, an order of magnitude faster than that for isolated thick filaments in the absence of actin. When a myofibril was imposed to shorten with a constant velocity by a piezoelectric actuator, the nucleotide displacement rate constant initially increased to 0.7 s(-1) with increasing shortening velocity and then declined with a further increase in shortening velocity. These results demonstrate that the displacement rates of Cy3-EDA-nucleotides bound to the cross-bridges in the contracting myofibril reflect a process that shows strain dependence.

Adenosine Triphosphatases↗
PubMed2006

Distinguishing the medicinal herb Oldenlandia diffusa from similar species of the same genus using fluorescence microscopy.

The light microscope is routinely used for microscopic identification of Chinese Materia Medica (CMM). However, the fluorescence microscope has not been used for this purpose until now. The CMM, Baihuasheshecao, mostly used as a component of herb tea, is a well known folk-medicine in China. According to the Chinese Pharmacopoeia, its source is the species Oldenlandia diffusa (Willd.) Roxb. Two other species of the same genus, namely O. corymbosa (L.) Lam and O. tenelliflora Bl., had been found in use as Baihuasheshecao. To find a quick and easy method to distinguish O. diffusa from these similar species of the same genus, the fluorescence microscope was used to investigate the fluorescence emission characteristics of the three tissues, which were compared with light microscopy images of the same material. The results showed that some tissues of the three herbs emit autofluorescence. Specially, the wall of endoderm cells of O. diffusa and O. tenelliflora emit autofluorescence, while similar tissue of O. corymbosa does not. Hence, fluorescence microscopy can be helpful in the identification of CMM.

Microscopy, Fluorescence↗
PubMed1979

Inhibition of fading in fluorescence microscopy of fixed cells.

Following Hirschfeld et al., dithionite was added to mounting media to inhibit fluorescence fading. Excellent response is reported for fluorescein, acridine orange, 33258 Hoechst, acriflavine, berberine (and ethidium bromide), but not for quinacrine.

Acridine Orange↗
PubMed1987

[Fluorescence microscopy detection of dermatophytes with Blankophor].

It is the first time that a fluorescent microscopic rapid-stain process is compared with the alkali method and with mycological culture examination. 810 skin scrapings primarily from horse, cat, and dog were available for analysis. The preparation of the samples for the fluorescent microscope test is easy and quick. The fluorescent stain solution keeps sufficiently long. When the slide preparations are looked at under the fluorescent microscope, the mycological elements light up and can easily be distinguished under survey enlargement. This leads to a considerable reduction of evaluation time. The microscopic proof of dermatophytes is considerably improved by the introduction of the fluorescent microscopic technique into mycological diagnosis, as this process is clearly superior to the alkali method. Dermatophytes are identified and finally evaluated after finishing the cultural analysis. Furthermore, the fluorescent microscopic proof of yeasts of the kind of Pityrosporum yeasts and of Demodex mites is possible. The not inconsiderable costs of the technical equipment may stand in the way of general and routine use of the fluorescent microscope for diagnosing dermatophytes. However, for laboratories with a large number of submitted skin scrapings, this process can be rated as a useful enrichment of their diagnostic potential.

Animals↗
PubMed1991

Illumination, wavelength selection, and detection in fluorescence microscopy.

The presently available devices for the illumination, changing of wavelengths, and detection of the resultant fluorescence of biological samples viewed in the light microscope have been described and compared. The optimal choice for illumination is a xenon arc lamp with a filter wheel wavelength selector. The optimal choice for an imaging detector is an intensified CCD (charge-coupled-device) camera. These combinations produce the most rapid, stable, and reproducible results when fluorescence measurements are made on living epithelial cells or isolated renal tubules. Techniques for the simultaneous acquisition of fluorescence and differential interference contrast (DIC) images have also been described and compared.

Animals↗
PubMed1998

Adsorption of complement, cytokines, and proteins by different dialysis membrane materials: evaluation by confocal laser scanning fluorescence microscopy.

The membranes tested in the present study were cellulose triacetate (CTA), polymethylmethacrylate (PMMA), and polyacrylonitrile (PAN). The adsorption by each membrane of albumin, IgG, C3a, interleukin-1beta (IL-1beta), interleukin-6 (IL-6), human neutrophil elastase (HNE), and tumor necrosis factor alpha (TNFalpha) was examined and semiquantitatively graded by confocal laser scanning fluorescence microscopy (CLSFM). After clinical use the dialyzers were treated with antibodies for these proteins and cytokines. Then the samples were incubated with fluorescein isothiocyanate-labeled anti-IgG antibody and observed by CLSFM. The changes in the blood levels of C3a and cytokines were also studied. In the CTA membrane, the adsorption of these substances, except for albumin and HNE, was less than in the synthetic membranes. The PAN membrane revealed the most abundant adsorption, especially for IL-1beta, IL-6, and TNFalpha. Although a marked elevation of C3a in the blood was observed in the CTA membrane, considerable adsorption was evident in the PMMA and the PAN membranes. Because the changes in the blood levels could be affected by membrane adsorption, both the blood levels and the adsorption of the biocompatibility parameters should be evaluated when membrane biocompatibility is discussed.

Acrylic Resins↗
PubMed2002

Exocytosis studies in a chromaffin cell-free system: imaging of single-vesicle exocytosis in a chromaffin cell-free system using total internal reflection fluorescence microscopy.

We have developed a system for the real-time study of regulated exocytosis in living, cultured bovine adrenal chromaffin cells (BCCs). Exocytosis was monitored by the use of total internal reflection fluorescence (TIRF) microscopy to image single large dense-core secretory vesicles (LDCVs). Fluorescent labeling of LDCVs was achieved either with the membrane-permeant weak base, acridine orange (AO), or by transduction of BCCs so as to express a fluorescent chimeric "cargo" protein that is targeted to LDCVs. In either case, exocytosis is visible by the disappearance of a vesicle accompanied by a bright flash as the fluorescent contents leave the acidic LDCV lumen, move towards the source of the evanescent wave, and disperse into the extracellular medium. Furthermore, for the first time, we have developed a broken-cell system for real-time imaging in BCCs, in which individual plated cells are mechanically "unroofed" with a jet of intracellular medium, leaving a membrane patch with docked vesicles on the coverslip. In this cell-free system, a subpopulation of docked granules undergoes exocytosis in response to calcium. This approach allows us direct experimental access to membrane-docked LDCVs in order to investigate the dependence of exocytosis on defined protein components and intracellular conditions at the single-vesicle level. In addition, this system can be used for a reconstitution analysis of the exocytosis machinery. Finally, we demonstrate the use of 2D+1 image analysis for visualizing single-vesicle exocytosis. We use this approach for a rapid analysis of larger numbers of imaged vesicles.

Acridine Orange↗